Polymer conjugates of Box-A of HMGB1 and Box-A variants of HMGB1

ABSTRACT

The present invention relates to novel polymer conjugates of polypeptide variants of the HMGB1 high affinity binding domain Box-A (HMGB1 Box-A) or of a biologically active fragment of HMGB1 Box-A. Further, the invention relates to novel polymer conjugates of polypeptide variants of the HMGB1 high affinity binding domain Box-A (HMGB1 Box-A). Moreover, the present invention concerns the use of said polymer conjugates of polypeptide molecules of HMGB1 Box-A to diagnose, prevent, alleviate and/or treat pathologies associated with extracellular HMGB1 and/or associated with an increased expression of RAGE.

This application is a divisional of U.S. Ser. No. 12/441,478 filed Mar.16, 2009, which is a 35 U.S.C. 371 National Phase Entry Application fromPCT/EP2007/08029 filed Sep. 14, 2007, which is a non-provisional of60/904,776 filed Mar. 5, 2007. This application claims the benefit ofEuropean Patent Application No. 06019362.0 filed on Sep. 15, 2006. Thedisclosures of which are incorporated herein in their entirety byreference.

DESCRIPTION

The present invention relates to novel polymer conjugates of the HMGB1high affinity binding domain Box-A (HMGB1 Box-A) or of a biologicallyactive fragment of HMGB1 Box-A. Further, the invention relates to novelpolymer conjugates of polypeptide variants of the HMGB1 high affinitybinding domain Box-A (HMGB1 Box-A) or of the biologically activefragments of HMGB1 Box-A. Moreover, the present invention concerns theuse of said polymer conjugates to diagnose, prevent, alleviate and/ortreat pathologies associated with extracellular HMGB1 and/or associatedwith an increased expression of RAGE.

The HMGB1 protein belongs to the family of high mobility group (HMG)proteins. HMG proteins, so-called due to their high electrophoreticmobility in polyacrylamide gels, are the most ubiquitous non-histoneproteins associated with isolated chromatin in eukaryotic cells. Theseproteins play a generalized “architectural” role in DNA bending,looping, folding and wrapping, since they either distort, bend or modifyDNA structures and complexes with transcription factors or histones(Andersson et al., 2002; Agresti et al., 2003; Degryse et al., 2003).The high mobility group 1 (HMGB1) protein is usually a nuclear factor,in particular a transcriptional regulatory molecule causing DNA bendingand facilitating the binding of several transcriptional complexes.

Structurally, the HMGB1 protein is a protein of approximately 25 kDawith a highly conserved sequence among mammals, whereby 2 out of 214amino acids have conservative substitutions in all mammalian species.HMGB1 is ubiquitously present in all vertebrate nuclei and in particularcan be found in fibroblasts, neurons, hepatocytes, glia and in cellsderived from hematopoietic stem cells, including monocytes/macrophages,neutrophils and platelets. The HMGB1 molecule has a tripartite structurecomposed of three distinct domains: two DNA binding domains called HMGBox-A and Box-B, and an acid carboxyl terminus, making it bipolarlycharged.

The two HMGB1 boxes are involved in the protein's function asnon-sequence-specific architectural DNA-binding elements, conferring theability to bind DNA into recognized distorted DNA structures andstabilizing nucleosome assembly, remodelling and sliding. Both the A-and B-HMG boxes are made up of highly conserved 84 amino acid residues,are strongly positively charged and are arranged in three α-heliceshaving a similar L-shaped fold. The long arm of the “L” contains theN-terminal extended strand and helix III (Andersson et al. 2002; Agrestiet al., 2003; Thomas, J. O. 2001), while the short arm comprises helicesI and II. Structure-function analysis reveals that the pro-inflammatorycytokine domain of HMGB1 is the B-Box and in particular the sequence ofits first 20 amino acids. The A-Box is an extremely weak agonist of theinflammatory cytokine release triggered by HMGB1 and competitivelyinhibits the pro-inflammatory activities of the B-Box and of the wholeprotein. Therefore, from a pharmacological point of view, the A-Box actsas an antagonist of the pathological conditions induced and/or sustainedby the B-Box and HMGB1.

The third domain, the carboxyl terminus or acidic tail, is extremelynegatively charged since it contains 30 repetitive aspartic and glutamicacid residues, and is linked to the boxes by a basic region of about 20residues. Mouse and rat HMGB1 differ from the human form by only twosubstitutions that are located in this continuous C-terminal stretch.

Besides its nuclear location and role as a transcription factorregulator, HMGB1 has also been found in the extracellular medium,actively released by activated cells of the immune systems (monocytesand macrophages) or passively released by damaged or necrotic cells(Andersson et al., 2002; Scaffidi et al., 2002; Bonaldi et al., 2002;Taniguchi et al., 2003; Friedman et al., 2003; Palumbo et al., 2004).

Extracellularly released HMGB1 acts as a potent cytokine and as anextremely potent macrophage-stimulating factor. HMGB1 acts directly bybinding to the cell membrane, inducing signaling and chemotaxis, havinga chemokine-like function (Yang et al., 2001) and further actingindirectly by up-regulating the expression and secretion ofpro-inflammatory cytokines. This makes extracellular HMGB1 protein apotent chemotactic and immunoregulatory protein which promotes aneffective inflammatory immune response. Furthermore, other proteinsbelonging to the family of HMG proteins, and which are able to bend DNA,are released together with HMGB1 in the extracellular medium. Theseproteins are inter alia HMGB2, HMGB3, HMG-1L10, HMG-4L and SP100-HMG.They share with HMGB1 highly homologous amino acid sequences. LikeHMGB1, they trigger/sustain inflammatory pathologies interacting withthe same receptors, leading to the same downstream pathways ofinteraction.

In healthy cells, HMGB1 migrates to the cytoplasm both by passive andactive transport. However, all cultured cells and resting monocytescontain the vast majority of HMGB1 in the nucleus, indicating that inbaseline conditions import is much more effective than export. Cellsmight transport HMGB1 from the nucleus by acetylating lysine residueswhich are abundant in HMGB1, thereby neutralizing their basic charge andrendering them unable to function as nuclear localization signals.Nuclear HMGB1 hyperacetylation determines the relocation of this proteinfrom the nucleus to the cytoplasm (in the fibroblasts, for example) orits accumulation into secretory endolysosomes (in activated monocytesand macrophages, for example) and subsequent redirection towards releasethrough a non-classical vesicle-mediated secretory pathway. HMGB1secretion by already activated monocytes is then triggered by bioactivelysophosphatidylcholine (LPC), which is generated later in theinflammation site from phosphatidylcholine through the action of thesecretory phospholipase sPLA2 produced by monocytes several hours afteractivation. Therefore, secretion of HMGB1 seems to be induced by twosignals (Bonaldi et al., 2003) and to take place in three steps: 1) atfirst, an inflammatory signal promotes HMGB1 acetylation and itsrelocation from the nucleus to the cytoplasm (step 1) and storage incytoplasmic secretory vesicles (step 2); then, a secretion signal(extracellular ATP or lysophosphatidylcholine) promotes exocytosis(third step) (Andersson et al., 2002; Scaffidi et al. 2002; Gardella etal., 2002; Bonaldi et al., 2003; Friedman et al., 2003).

Released HMGB1 has been identified as one of the ligands binding to theRAGE receptor. This receptor is expressed in most cell types, and at ahigh level mainly in endothelial cells, in vascular smooth muscle cells,in monocytes and macrophages and in mononuclear phagocytes. Recognitioninvolves the C-terminal of HMGB1. The interaction of HMGB1 and RAGEtriggers a sustained period of cellular activation mediated by RAGEupregulation and receptor-dependent signaling. In particular, theinteraction of HMGB1 and RAGE activates several intracellular signaltransduction pathways, including mitogen-activated protein kinases(MAPKs), Cdc-42, p21ras, Rac and the nuclear translocation factor κB(NF-κB), the transcription factor classically linked to inflammatoryprocesses (Schmidt et al., 2001).

According to several experimental evidences, released HMGB1 may alsointeract with receptors belonging to one or more subclass(es) of thefamily of the Toll-like receptors. Further, HMGB1 may also interact withthe functional N-terminal lectin-like domain (D1) of thrombomodulin. Dueto the ability of the functional D1 domain of thrombomodulin tointercept and bind circulating HMGB1, the interaction with the RAGEreceptors and the Toll-like receptors is prevented.

When released in vivo, HMGB1 is an extremely potent cytokine and apotent macrophage-stimulating factor. In fact, like other cytokinemediators of endotoxemia, HMGB1 activates in vitro a cascade of multiplepro-inflammatory cytokines (TNF, IL-1α, IL-1β, IL-1Ra, IL-6, IL-8,MIP-1α and MIP-1β) from human macrophages. Therefore, HMGB1 acts as alate mediator during acute inflammation and participates in an importantway in the pathogenesis of systemic inflammation after the earlymediator response has been resolved.

Moreover, the observed RAGE upregulation in proinflammatory environmentsand the proved increased expression of this receptor in a variety ofacute and chronic inflammatory diseases provide support for RAGE as anattractive target for future medical interventions related toinflammation.

The observed pro-inflammatory effects of HMGB1 in vitro and thecorrelation between circulating HMGB1 levels and the development of thepathogenic sequence of systemic inflammation in vivo indicate thattherapeutically targeting of this cytokine-like molecule should be ofrelevant clinical value, suggesting novel therapeutic approaches by a“late” administration of (selective) antagonists/inhibitors of theextracellular activities of HMGB1.

Therefore, several attempts were performed in order to block thisextracellular HMGB1 chemo-cytokine protein. Several important approacheswere addressed to the administration of antibodies against HMGB1, ofantibodies against HMGB1 fragments (for example HMGB1 Boxes) ofantibodies to RAGE, of soluble RAGE (sRAGE), of ethyl pyruvate (Czura etal., 2003; Lotze et al., 2003) and N-terminal lectin-like domain (D1) ofthrombomodulin.

HMGB1 A-Box, one of the two DNA-binding domains in HMGB1, has beenidentified as a specific antagonist of HMGB1: highly purifiedrecombinant A-Box has protected mice from lethal experimental sepsiseven when initial treatment has been delayed for 24 hours afterpathology induction, further suggesting that HMGB1 antagonists may beadministered successfully in a clinically relevant window wider than theone used for other known cytokines (Yang et al., 2004).

Structural function analysis of HMGB1-truncated mutants has revealedthat the A-Box domain of HMGB1 competitively displaces the saturablebinding of HMGB1 to macrophages, specifically antagonizing HMGB1activities. As has been already seen with the protective activity ofanti-HMGB1 antibodies, the administration of the A-Box rescues mice fromsepsis even when treatment has been initiated as late as 24 hours aftersurgical induction of sepsis (Yang H. et al., 2004). HMGB1 antagonistsor inhibitors selected from the group of antibodies or antibodyfragments that bind to an HMGB1 protein, HMGB1 gene antisense sequencesand HMGB1 receptor antagonists are known from U.S. Pat. No. 6,468,533,WO 02/074337 and US 2003/0144201.

A promising attempt for inhibiting and/or antagonising the extracellularHMGB1 chemo-cytokine protein is therefore based on the experimentalevidence that the two high affinity binding domains for DNA, i.e. HMGB1Box-A and HMGB1 Box-B, which are present in the HMGB1 molecule, have twoopposing roles in the protein released in the extracellular space. Themain activity of HMGB1 Box-B is to mediate the pro-inflammatoryactivities attributed to the HMGB1 protein. On the other hand, HMGB1Box-A acts as an antagonist competing with the pro-inflammatory activityof the Box-B domain.

The patent application WO 2006/024547 discloses polypeptide variants ofthe HMGB1 Box-A, or of biologically active fragments of HMGB1 Box-A,which are obtained through systematic mutations of single amino acids ofthe wild-type HMGB1 Box-A protein. Therefore, WO 2006/024547 providesnew agents as selective inhibitors and/or antagonists of extracellularHMGB1 and their use to prevent, alleviate and/or treat the broadspectrum of pathological conditions associated and induced by theextracellular HMGB1 chemokine and/or by the cascade of multipleinflammatory cytokines caused by the extracellular release of the HMGB1chemokine proteins.

The efficacy of administration of synthetic protein drugs may behampered in vivo by factors such as solubility at physiological pH,rapid elimination by glomerulal filtration, cellular clearance andmetabolism as well as readily absorption. The efficacy of oraladministration is, for example, hampered since proteins are digested iftaken orally. The efficacy of systemic administration on the other handis hampered since proteins under 65-70 kDa are cleared rapidly from thebody. In many cases, such disadvantageous effects lead to reducedpatient compliance and to reduced drug efficacy preventing an effectivetherapeutic use of such protein agents.

A successful strategy for improving the efficacy and the duration of theprotein agent effects and for reducing potential toxicological effectsis the covalent binding of a biologically active protein agent todiverse polymers. One of the polymers that is most often used in the artfor improving the pharmacologic and toxicologic properties of an activeagent is polyethyleneglycol, PEG in short. Polyethyleneglycol (PEG)polymers are amphiphilic, non-toxic and immunologically inert and can beconjugated to pharmaceuticals to manipulate many of the pharmacokineticand toxicologic properties.

In the art, many covalent modification of therapeutic useful proteinswith polyethyleneglycol (PEG) are reported. Covalent attachment of PEGto a protein (“pegylation”) is useful in order to extend the circulationhalf life of proteins, since it increases the proteins effective sizeand reduces it rate of clearance from the body. Moreover, PEGmodification of a protein increases the protein solubility, stabilityand decreases the protein immunogenicity.

The problem underlying the present invention was therefore the provisionof novel therapeutically useful protein agents, which act as selectiveantagonist and/or inhibitors of extracellular HMGB1. The scope of thepresent invention was therefore to exploit the peculiar characteristicsof some polymers, in particular of PEG, in order to develop newadministration forms of the HMGB-1 high affinity binding domain Box-A(HMGB1 Box-A), which show the same if not an even higher pharmacologicalactivity and moreover, an improved pharmacokinetic and toxicologicperformance in comparison to the non-conjugation HMGB1 Box-A polypeptideand which permit to achieve the best availability of HMGB1 Box-A or of abiologically active fragment thereof in various possible administrationroutes.

The present invention is therefore directed to a novel polymer conjugateof the human and/or non-human wild type HMGB1 high affinity bindingdomain Box-A (HMGB1 Box-A) or of a biological active fragment of HMGB1Box-A. The amino acid sequence of human HMGB1 Box-A is shown in SEQ IDNO:1. A preferred non-human HMGB1 Box-A is the Anopheles gambia HMGB1Box-A, the sequence of which is shown in SEQ ID NO:301.

A further aspect of the present invention is directed to a polymerconjugate of a polypeptide variant of the human and/or non-human HMGB1high affinity binding domain Box-A or of a biologically active fragmentof HMGB1 Box-A, whereby the amino acid sequence of said polypeptidevariant differs from the amino acid sequence of the wild type HMGB1Box-A by the mutation of one or more single amino acids.

In the context of the present invention, “HMGB1” includes thenon-acetylated form or/and the acetylated form of HMGB1. Likewise,“HMGB1 homologous proteins” include the non-acetylated form or/and theacetylated form of HMGB1 homologous proteins. Preferred HMGB1 homologousproteins are HMGB2, HMGB3, HMG-1L10, HMG-4L or/and SP100-HMG.

The novel polymer conjugates of the present invention show an increasedwater solubility, an improved pharmaceutical manageability, an improvedpharmacokinetic and bioavailability and/or a decreased toxicity and/orimmunogenicity in comparison to the non-conjugated HMGB1 polypeptide orpolypeptide variant. Moreover, it was surprisingly found that theconjugation of the HMGB1 Box-A polypeptide or polypeptide variant and/orfragment does not alter the biological activity of the protein.

The polymer moiety according to the present invention has to bebiocompatible, can be of natural or semi-synthetic or synthetic originand can have a linear or branched structure. Exemplary polymers includewithout limitation polyalkylene glycols, polyalkylene oxides,polyacrylic acid, polyacrylates, polyacrylamide or N-alkyl derivativesthereof, polymethacrylic acid, polymethacrylates, polyethylacrylic acid,polyethylacrylates, polyvinylpyrrolidone, poly(vinylalcohol),polyglycolic acid, polylactic acid, poly(lactic-co-glycolic) acid,dextran, chitosan, polyaminoacids.

In a very preferred embodiment of the present invention, the polymer ispolyethylene glycol (PEG) or polyethylene glycol, wherein the terminalOH group can optionally be modified, e.g. with C₁-C₅ alkyl groups orC₁-C₅ acyl groups, preferably with C₁-, C₂- or C₃ alkyl groups or C₁-,C₂- or C₃ acyl groups. Preferably, the modified polyethylene glycol ismethoxy-polyethylene-glycol (mPEG).

The polymer used according to the present invention has a molecularweight ranking from 100 to 100,000 Da, preferably from 5,000 to 50,000Da. In a very preferred embodiment of the invention, the polymer is PEG,which preferably has a terminal OH and/or methoxy group, with amolecular weight ranking from 10,000 to 40,000 Da, and preferably from20,000 to 40,000 Da. In the most preferred embodiment, a PEG, whichpreferably has a terminal OH and/or methoxy group, with an averagemolecular weight of 20,000 Da or of 40,000 Da is used in the presentinvention.

The polymer moiety of the polymer conjugate of the invention isconjugated to the HMGB1 polypeptide or polypeptide variant by a covalentchemical bond in order to provide a stable conjugate.

Preferred conjugation sites on the HMGB1 Box-A moiety are selected froma lysine, cysteine, histidine, arginine, tyrosine, serine, threonine,aspartate and glutamate residue or from the N-terminal amino group ofthe protein moiety.

The polymer conjugate of the present invention may be mono-, di- andmulti-pegylated conjugates. Preferably, the polymer conjugates of theinvention are mono-pegylated.

The polymer moiety is usually covalently linked to the HMGB1 Box-Amoiety through a linker group. In particular, in the context of thepresent invention the term linker group means a group which is obtainedby the chemical reaction between polypeptide moiety and polymer moiety.The linker group can be any residue known to those skilled in the art ofpolymer conjugation, obtained by the reaction of the active group on theamino acid residue of the HMGB1 Box-A moiety and the polymer or thepolymer activated by a reactive group. Exemplary linker groups includewithout limitation alkylene, amine, amide, carbamate, carboxylate,carbonyl, ester, ether, thioether and disulfide groups. Preferably, thelinker group is an amine bond, which is obtained by the reaction of theN-terminal amino acid residue with the polymer moiety activated with analdehyde reactive group and subsequent reduction.

Moreover, the linker group may optionally contain one or more spacergroups. In the context of the present invention, a spacer group isdefined as a bifunctional group, having on both termini a reactivefunctional end-group. With the one reactive end-group, the spacer reactswith the polymer moiety or with the reactive group on the polymermoiety. With the further functional group on the other terminus, thespacer group binds to the functional group on the amino acid residue ofthe HMGB1 Box-A moiety. Suitable spacer groups are known to thoseskilled in the art. Examples of spacer groups include, but are notlimited to hetero-, bi-functional small molecules or polymer. Forexample, the spacer group may be represented by bifunctional C₆-C₁₂alkyl groups or heterobifunctional alkyl groups containing from 1-3heteroatoms selected from N, S and O or an intermediary shortbifunctional PEG chain.

Covalent attachment of the polymer to the HMGB1 Box-A moiety to obtainthe polymer conjugate of the invention may be accomplished by knownchemical synthesis techniques. For example, in one exemplary embodimentof the present invention, the polymer conjugation can be accomplished byreacting a N-hydroxy succinimide polymer (es. NHS-PEG) with the freeamine groups on the amino acid residues, preferably on the lysineresidues, or at the N-terminal amino acid of the HMGB1 Box-Apolypeptide. Alternatively, the polymer conjugation is achieved byreaction of a PEG aldehyde to the N-terminus of the polypeptide byreductive amination. Further, the polymer conjugates can also beobtained by reacting a PEG-maleimide to a Cys residue of the HMGB1 Box-Apolypeptide or polypeptide variant.

In the context of the present invention, the term “HMGB1 Box-A moiety”indicates within the polymer conjugate compound the polypeptide moiety.Hence, this term refers to the wild-type HMGB1 Box-A and to biologicallyactive fragments thereof as well as to polypeptide variants of HMGB1Box-A and of biologically active fragments thereof.

In the context of the present invention, where reference is made to theterm “HMGB1 Box-A or amino acid sequence of HMGB1 Box-A”, it is referredto both human and non-human HMGB1 Box-A. In a preferred embodiment ofthe present invention, the HMGB1 Box-A moiety is derived from the wildtype of human HMGB1 Box-A protein and from the wild type of Anophelesgambia HMGB1 Box-A protein.

“Biologically active fragments of HMGB1 Box-A” as used herein are meantto encompass parts of the known wild type HMGB1 Box-A protein, for whichat least one of the biological activities of the corresponding matureprotein is still observable when known tests are being used. Preferably,a fragment of the mature protein is considered as biologically active ifan antagonist activity with respect to the pro-inflammatory activity ofthe HMGB1 B-Box and the HMGB1 protein as a whole can be determined.Biologically active fragments of native HMGB1 Box-A are fragments of atleast 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75 or 80 amino acids.Preferred biologically active fragments of native HMGB1 Box-A used inthe context of the present invention comprises fragments of at least 77or of at least 54 amino acids, respectively.

The term “mutation” as used in the context of the present invention canbe understood as substitution, deletion and/or addition of single aminoacid in the target sequence. Preferably, the mutation of the targetsequence in the present invention is a substitution. The substitutioncan occur with different genetically encoded amino acid or bynon-genetically encoded amino acids. Examples for non-geneticallyencoded amino acids are homocystein, hydroxyproline, ornithin,hydroxylysine, citrulline, carnitine, etc.

In a most preferred embodiment of the present invention, the polypeptidevariants of HMGB1 Box-A or of a biologically active fragment thereof areobtained by using a directed evolution process, which technology isextensively described in WO 2004/7022593 and in several further patentapplications (PCT/FR00/03503, PCT/FR01/01366, U.S. Ser. No. 10/022,249,U.S. Ser. No. 10/022,390, U.S. Ser. No. 10/375,192, U.S. 60/409,898,U.S. 60/457,135, U.S. 60/410,258 and U.S. 60/410,263), all in the nameof Nautilus Biotech S.A. (Paris, France), which are herein incorporatedby reference.

The polypeptide variants of the present invention obtained by usingdirected evolution technology are mutant proteins which differ from theamino acid sequence of the wild type HMGB1 Box-A by the mutation of oneor more single amino acid. In a very preferred embodiment of the presentinvention, only one amino acid replacement occurs on the sequence of thenative protein. It is, however, encompassed by the subject of thepresent invention that the native protein can be further optimised byreplacement of a plurality, e.g. two or more, of amino acidreplacements. The modified polypeptide variants can therefore differfrom the wild type protein sequence by amino acid replacements on 1-10,preferably 2, 3, 4, 5 and 6 different amino acid target positions.

In particular, the very preferred polypeptide variants of HMGB1 Box-A orof a biologically active fragment thereof used as HMGB1 Box-A moiety ofthe polymer conjugates of the invention are those described in theapplication WO 2006/024547.

Accordingly, in one preferred embodiment of the invention, the HMGB1Box-A moiety of the polymer conjugate is derived starting from humanHMGB1 Box-A. In particular, one group of polypeptide variants is derivedfrom single mutations introduced into the full-length amino acidsequence (84 amino acids) from Human HMGB1 Box-A (SEQ ID NO:1) (FIG. 1a). These preferred polypeptide variants are defined in sequences SEQ IDNos:2-116 (FIG. 1b ).

Other preferred polypeptide variants are obtained starting frombiologically active fragments of human HMGB1 Box-A of 77 amino acids(SEQ ID NO:117) (FIG. 2a ) and 54 amino acids (SEQ ID NO:223) (FIG. 3a), respectively. The polypeptide variants of Box-A of human HMGB1fragment of 77 amino acids are defined in sequences SEQ ID NOs:118 to222 (FIG. 2b ). The polypeptide variants of Box-A of human HMGB1fragment of 54 amino acids are defined in sequences SEQ ID NOs:224 to300 (FIG. 3b ).

In a further preferred embodiment of the invention, the HMGB1 Box-Amoiety of the polymer conjugate is derived starting from Anophelesgambia HMGB1 Box-A. In particular, one group of polypeptide variants isderived from single mutations introduced into the full-length amino acidsequence (84 amino acids) from Anopheles gambia HMGB1 Box-A (SEQ IDNO:301) (FIG. 4a ). These polypeptide variants are identified in thesequences SEQ ID Nos:302 to 418 (FIG. 4b ). Other preferred polypeptidevariants are generated starting from biologically active fragments ofAnopheles gambia HMGB1 Box-A of 77 amino acids (SEQ ID NO:419) (FIG. 5a) and 54 amino acids (SEQ ID NO:529) (FIG. 6a ), respectively. Thepolypeptide variants of Box-A of HMGB1 fragment of 77 amino acids aredefined in sequences SEQ ID Nos:420 to 528 (FIG. 5b ). The polypeptidevariants of Box-A of HMGB1 Anopheles gamble (XP_311154) fragment of 54amino acids are defined in sequences SEQ ID Nos:530 to 610 (FIG. 6b ).

In order to identify the most preferred polypeptide variants of HMGB1Box-A used as HMGB1 Box-A moiety of the polymer conjugates of theinvention, studies have been conducted to determine the polypeptidevariants which show both a similar or even improved activity and anincreased protease resistance compared to the wild-type HMGB1 Box-Aprotein. For this purpose, the activity of Box-A polypeptide variants ofthe human HMGB1 Box-A of SEQ ID NO:1 in inhibiting HMGB1-induced NIH/3T3cell migration was determined in chemotaxis assays in comparison to theantagonistic activity of human HMGB1 Box-A wild-type itself (Example 1and FIGS. 7.1 to 7.9). Moreover, for those polypeptide variants, whichshow a similar or even higher antagonistic activity than the nativeHMGB1 Box-A protein of SEQ ID NO:1, the in vitro resistance to proteasedigestion was determined by incubation of each of these polypeptidevariants, with a mixture of trypsin, α-chymotrypsin, endoproteinaseAsp-N and endoproteinase Glu-C (sigma). This protease resistance test isdescribed in Example 2 and the results of protease resistance profile ofsaid variants in comparison to native HMGB1 Box-A are shown in FIGS. 9.1to 9.67.

From the results it can be gathered that the preferred polypeptidevariants of HMGB1 Box-A useful as HMGB1 Box-A moiety of the polymerconjugate of the present invention are those variants which show asimilar or higher antagonistic activity together with an increasedprotease resistance. In particular, the preferred polypeptide variantsare the polypeptides of SEQ ID NOs: 33, 35, 37-39, 42-45, 47-49, 52, 55,57, 59, 62, 64, 67, 69 and 104. Among these preferred polypeptidevariants, the most preferred variants are those defined in SEQ IDNos:45, 49, 52, 55, 59, 64 and 67. These very preferred polypeptidevariants show a dramatically improved proteinase resistance profilecompared with the wild-type human HMGB1 Box-A of SEQ ID NO:1 (cf.results of Example 2).

It is noted that the amino acids which occur in the various amino acidsequences appearing herein are identified according to their knownone-letter code abbreviations. It should be further noted that all aminoacid residue sequences represented herein by their one-letterabbreviation code have a left-to-right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus.

In the present invention, it was surprisingly found that the abovedescribed polymer conjugates exhibit an improved pharmacokinetic andtoxicologic performance, leading to an improved bioavailability comparedto the non-conjugated HMGB1 Box-A polypeptide moiety. A particularadvantageous effect of the polymer conjugation of the HMGB1 Box-Apolypeptides and variants thereof, and in particular of the pegylationof these polypeptides, in comparison with the non-conjugated form, isthe increase of the hydrodynamic volume of the proteins. This leads to asignificant and unexpected improvement of the pharmacokinetic propertiesof the conjugated compounds due to the avoidance of renal clearance,i.e. reduction of glomerular filtration.

Moreover, the polymer conjugates of the invention exhibit increasedresistance to the proteolytic activity of proteases and/or peptidases,in particular exhibit an increased resistance to the proteolyticactivity of the human proteases and/or peptidases, in particular of thehuman serum proteases and/or human gastro-intestinal proteases orpeptidases.

In particular, the resistance to proteolysis is at least 10%, 20%, 30%,40%, 50%, 70%, 80%, 90%, 95% or higher compared to the non-conjugatedHMGB1 Box-A. Protease resistance was measured at different timepoints(between 5 minutes and 8 hours) at 25° C. after incubation of 20 μg ofBox-A wild type or variants with a mixture of proteases at 1% w/w oftotal proteins. The mixture of the proteases was prepared freshly ateach assay from stock solutions of endoproteinase Glu-C (SIGMA) 200μg/ml; trypsin (SIGMA) 400 μg/ml and α-chymotrypsin (SIGMA) 400 μg/ml.After protease incubation the reaction was stopped adding 10 μl ofanti-proteases solution (Roche) and the samples were stored at −20° C.for the biological activity assay.

As a consequence of the increased stability due to the increasedresistance to proteases activity, the polymer conjugates of the presentinvention also exhibit a longer half-life in body fluids compared to thenon-conjugated HMGB1 Box-A. In particular, the half-life in serum and/orin blood is increased, whereby an increase of at least 10 minutes, 20minutes, 30 minutes, 60 minutes or even longer, compared to thenon-conjugated HMGB1 Box-A is observed.

Due to the increase of the hydrodynamic volume of the proteins and alsodue to an increased resistance to proteolysis and thus the higherstability, the polymer conjugates of the invention also exhibit improvedtherapeutic and biological properties and activity. In fact, they show amore favorable pharmacokinetic and pharmacodynamic profile thannon-conjugated HMGB1 Box-A protein and protein variants.

The invention is therefore directed to the use of the above-mentionedpolymer conjugates of HMGB1 Box-A as an active agent in a medicament.

A still further aspect of the invention is hence the use of theinventive polymer conjugates for the manufacture of a medicament for theprevention and/or treatment of extracellular HMGB1-associatedpathologies or pathologies associated with the HMGB1 homologousproteins. In particular, the HMGB1 associated pathologies arepathologies which are mediated by a multiple inflammatory cytokinecascade.

The broad spectrum of pathological conditions induced by theHMGB1-chemokine and by the HMGB1-induced cascade of inflammatorycytokines are grouped in the following categories: inflammatory disease,autoimmune disease, systemic inflammatory response syndrome, reperfusioninjury after organ transplantation, cardiovascular affections, obstetricand gynecologic disease, infectious (viral and bacterial) disease,allergic and atopic disease, solid and non-solid tumor pathologies,transplant rejection diseases, congenital diseases, dermatologicaldiseases, neurological diseases, cachexia, renal diseases, iatrogenicintoxication conditions, metabolic and idiopathic diseases.

HMGB1-associated pathologies according to the present invention arepreferably pathological conditions mediated by activation of theinflammatory cytokine cascade. Non limiting examples of conditions whichcan be usefully treated using the present invention include the broadspectrum of pathological conditions induced by the HMGB1-chemokine andby the HMGB1-induced cascade of inflammatory cytokines grouped in thefollowing categories: restenosis and other cardiovascular diseases,reperfusion injury, inflammation diseases such as inflammatory boweldisease, systemic inflammation response syndrome, e.g. sepsis, adultrespiratory distress syndrome, etc, autoimmune diseases such asrheumatoid arthritis and osteoarthritis, obstetric and gynaecologicaldiseases, infectious diseases, atopic diseases, such as asthma, eczema,etc, tumor pathologies, e.g. solid or non-solid tumor diseasesassociated with organ or tissue transplants, such as reperfusioninjuries after organ transplantation, organ rejection andgraft-versus-host disease, congenital diseases, dermatological diseasessuch as psoriasis or alopecia, neurological diseases, ophthalmologicaldiseases, renal, metabolic or idiopathic diseases and intoxicationconditions, e.g. iatrogenic toxicity and Behçet disease, wherein theabove diseases are caused by, associated with and/or accompanied byHMGB1 protein release.

In particular, the pathologies belonging to inflammatory and autoimmunediseases include rheumatoid arthritis/seronegative arthropathies,osteoarthritis, inflammatory bowel disease, Crohn's disease, intestinalinfarction, systemic lupus erythematosus, iridoeyelitis/uveitis, opticneuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,systemic sclerosis and scleroderma. Systemic inflammatory responseincludes sepsis syndrome (including gram positive sepsis, gram negativesepsis, culture negative sepsis, fungal sepsis, neutropenic fever,urosepsis, septic conjunctivitis), meningococcemia, trauma hemorrhage,hums, ionizing radiation exposure, acute and chronic prostatitis, acuteand chronic pancreatitis, appendicitis, peptic, gastric and duodenalulcers, peritonitis, ulcerative, pseudomembranous, acute and ischemiccholitis, diverticulitis, achalasia, cholangitis, cholecystitis,enteritis, adult respiratory distress syndrome (ARDS). Reperfusioninjury includes post-pump syndrome and ischemia-reperfusion injury.Cardiovascular disease includes cardiac stun syndrome, myocardialinfarction and ischemia, atherosclerosis, thrombophlebitis,endocarditis, pericarditis, congestive heart failure and restenosis.Obstetric and gynecologic diseases include premature labour,endometriosis, miscarriage, vaginitis and infertility. Infectiousdiseases include HIV infection/HIV neuropathy, meningitis, B- andC-hepatitis, herpes simplex infection, septic arthritis, peritonitis, E.coli 0157:H7, pneumonia epiglottitis, haemolytic uremicsyndrome/thrombolytic thrombocytopenic purpura, candidiasis, filariasis,amebiasis, malaria, Dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, Lyme disease, influenza A,Epstein-Barr Virus, Cytomegalovirus, viral associated hemiaphagocyticsyndrome, viral encephalitis/aseptic meningitis. Allergic and atopicdisease include asthma, allergy, anaphylactic shock, immune complexdisease, hay fever, allergic rhinitis, eczema, allergic contactdermatitis, allergic conjunctivitis, hypersensitivity pneumonitis.Malignancies (liquid and solid tumor pathologies) include ALL, AML, CML,CLL, Hodgkin's disease, non Hodgkin's lymphoma, Kaposi's sarcoma,colorectal carcinoma, nasopharyngeal carcinoma, malignant histiocytosisand paraneoplastic syndrome/hypercalcemia of malignancy. Transplantdiseases include organ transplant rejection and graft-versus-hostdisease. Congenital disease includes cystic fibrosis, familialhematophagocytic lymphohistiocytosis and sickle cell anemia.Dermatologic disease includes psoriasis, psoriatic arthritis andalopecia. Neurologic disease includes neurodegenerative diseases(multiple sclerosis, migraine, headache, amyloid-associated pathologies,prion diseases/Creutzfeld-Jacob disease, Alzheimer and Parkinson'sdiseases, multiple sclerosis, amyotrophic emilateral sclerosis) andperipheral neuropathies, migraine, headache. Renal disease includesnephrotic syndrome, hemodialysis and uremia. Iatrogenic intoxicationcondition includes OKT3 therapy, Anti-CD3 therapy, Cytokine therapy,Chemotherapy, Radiation therapy and chronic salicylate intoxication.Metabolic and idiopathic disease includes Wilson's disease,hemochromatosis, alpha-1 antitrypsin deficiency, diabetes and diabetescomplications, weight loss, anorexia, cachexia, obesity, Hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation and primary biliary cirrhosis. Opthalmological diseaseinclude glaucoma, retinopathies and dry-eye. A miscellanea of otherpathologies comprehends: multiple organ dysfunction syndrome, musculardystrophy, septic meningitis, atherosclerosis, epiglottitis, Whipple'sdisease, asthma, allergy, allergic rhinitis, organ necrosis, fever,septicaemia, endotoxic shock, hyperpyrexia, eosinophilic granuloma,granulomatosis, sarcoidosis, septic abortion, urethritis, emphysema,rhinitis, alveolitis, bronchiolitis, pharyngitis, epithelial barrierdysfunctions, pneumoultramicropicsilicovolcanoconiosis, pleurisy,sinusitis, influenza, respiratory syncytial virus infection,disseminated bacteremia, hydatid cyst, dermatomyositis, burns, sunburn,urticaria, warst, wheal, vasulitis, angiitis, myocarditis, arteritis,periarteritis nodosa, rheumatic fever, celiac disease, encephalitis,cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia,iatrogenic complications/peripheral nerve lesions, spinal cord injury,paralysis, uveitis, arthriditis, arthralgias, osteomyelitis, fasciitis,Paget's disease, gout, periodontal disease, synovitis, myastheniagravis, Goodpasture's syndrome, Behçets's syndrome, ankylosingspondylitis, Barger's disease, Retier's syndrome, bullous dermatitis(bullous pemphigoid), pemphigous and pemphigous vulgaris and alopecia.

In a further preferred embodiment, the polymer compounds of theinvention are used as active agents for the prevention, alleviationand/or treatment of RAGE-related pathologies. RAGE-related pathologiesare defined as pathological states associated with an increasedexpression of RAGE.

RAGE (Receptor for Advanced Glycation End-products) is a multi-ligandmember of the immunoglobulin superfamily of cell surface molecules. Itis composed of three immunoglobulin-like regions (one V-typeimmunoglobulin domain followed by two C-type immunoglobulin domain), atransmembrane domain and a highly charged short cytosolic tail that isessential for post-RAGE signalling. RAGE was first identified in 1992 asa binding target for AGEs, non-enzymatically glycosylated and oxidatedproteins which accumulate in vascular tissue in aging and at anaccelerated rate in diabetes. RAGE is expressed on a wide set of cells,including endothelial cells, smooth muscle cells, mononuclear phagocytesand neurons. While it is present at high levels during development,especially in the central nervous system, its levels decline duringmaturity.

As reported above, RAGE was the first receptor identified forextracellular HMGB1. HMGB1 binding on the cell surface induces thetranscriptional upregulation of RAGE. Examples of RAGE-relatedpathologies are diabetes and disorders associated with diabetes such asdiabetic vasculopathy, neuropathy, retinopathy and other disorders,including Alzheimer's disease and immune/inflammatory reactions of thevessel walls. A very preferred example of RAGE-related pathologies inthis context is diabetes of type I and/or of type II.

In a further aspect of the invention, the use of the polymer conjugatesHMGB1 Box-A described above is in combination with a further activeagent.

The further agent is preferably an agent capable of inhibiting an earlymediator of the inflammatory cytokine cascade. Preferably, this furtheragent is an antagonist or inhibitor of a cytokine selected from thegroup consisting of TNF, IL-1α, IL-1β, IL-Ra, IL-6, IL-8, IL-10, IL 13,IL-18, IFN-γ MIP-1α, MIF-1β, MIP-2, MIF and PAF.

The further agent used in combination with the polymer conjugate, mayalso be an inhibitor of RAGE, e.g. an antibody directed to RAGE, anucleic acid or nucleic acid analogue capable of inhibiting RAGEexpression, e.g. an antisense molecule, a ribozyme or a RNA interferencemolecule, or a small synthetic molecule antagonist of the interaction ofHMGB1 with RAGE, preferably of the interaction of the non-acetylatedor/and acetylated form of HMGB1 with RAGE, or soluble RAGE (sRAGE). Theantibody to RAGE is preferably a monoclonal antibody, more preferably achimeric or humanised antibody or a recombinant antibody, such as asingle chain antibody or an antigen-binding fragment of such anantibody. The soluble RAGE analog may be optionally present as a fusionprotein, e.g. with the Fc domain of a human antibody. The smallsynthetic molecular antagonist of the HMGB1 interaction with RAGEpreferably has a molecular weight of less than 1000 Dalton. The smallsynthetic molecular antagonist preferably inhibits the interaction ofRAGE with the non-acetylated form or/and with the acetylated form ofHMGB1 and with the non-acetylated form or/and with the acetylated formof HMGB1 homologous proteins, particularly HMGB2, HMGB3, HMG-1L10,HMG-4L or/and SP100-HMG.

The further agent used in combination with the polymer conjugate, mayalso be an inhibitor of the interaction of a Toll-like receptor (TLR),e.g. of TLR2, TLR4, TLR7, TLR8 or/and TLR9, with HMGB1, which inhibitoris preferably a monoclonal or polyclonal antibody, a nucleic acid ornucleic acid analogue capable of inhibiting TLR expression, e.g. anantisense molecule, a ribozyme or a RNA interference molecule, or asynthetic molecule preferably having a size of less than 1000 Dalton.The inhibitor may be a known inhibitor of a Toll-like receptor, inparticular of TLR2, TLR4, TLR7, TLR8 or/and TLR9. The inhibitorpreferably inhibits the interaction of the Toll-like receptor with thenon-acetylated form or/and the acetylated form of HMGB1 and with thenon-acetylated form or/and with the acetylated form of HMGB1 homologousproteins, in particular HMGB2, HMGB3, HMG-1L10, HMG-4L or/and SP100-HMG.

In still another embodiment, the further agent is the functionalN-terminal lectin-like domain (D1) of thrombomodulin. The D1 domain ofthrombomodulin is able to intercept the non-acetylated form and/or theacetylated form of released HMGB1 and of released HMGB1 homologousproteins, in particular HMGB2, HMGB3, HMG-1L10, HMG-4L or/and SP100-HMG,preventing thus their interaction with RAGE and Toll-like receptors. TheD1 domain of thrombomodulin may be native or mutated in order to make itresistant to proteases.

The further agent may also be a synthetic double-stranded nucleic acidor nucleic acid analogue molecule with a bent shape structure,particularly a double-stranded bent DNA, PNA or DNA/PNA chimera orhybrid or a double-stranded cruciform DNA, PNA or DNA/PNA chimera orhybrid structure, capable of binding to the HMGB1 protein. Preferrednucleic acids and nucleic analogue molecules are disclosed in a co-ownedand co-pending international patent application No. PCT/EP2005/007198filed on 4 Jul. 2005 (claiming the priority of U.S. provisionalapplication No. 60/584,678 filed on 2 Jul. 2004), which are incorporatedherein by reference. The synthetic double-stranded nucleic acid ornucleic acid analogue molecule with a bent shape structure is preferablycapable of binding to the non-acetylated or/and to the acetylated formof HMGB1 and the non-acetylated or/and the acetylated form of HMGB1homologous proteins, in particular HMGB2, HMGB3, HMG-1L10, HMG4L or/andSP100-HMG.

In a still further embodiment, the further agent used in combinationwith the polymer conjugate is K-252a or/and a salt or derivative thereofor a polymer conjugate of K-252a or/and of a derivative thereof. The useof K-252a or polymer conjugates of K-252a and derivatives thereof isdisclosed in a co-owned and co-pending international patent applicationNo. PCT/EP2005/008258 and filed on 25 Aug. 2005, which is hereinincorporated by reference.

Therefore, a further aspect of the present invention is a pharmaceuticalcomposition comprising an effective amount of at least one of thepolymer conjugates of HMGB1 Box-A polypeptide or polypeptide variant ora biologically active fragment thereof as an active ingredient for thetreatment of HMGB1-associated pathologies and pharmaceuticallyacceptable carriers, diluents and/or adjuvants. The pharmaceuticalcomposition of the present invention is preferably suitable for thetreatment of pathologies associated with the non-acetylated or/and theacetylated form of HMGB1 and/or of HMGB1 homologous proteins. In afurther preferred embodiment, the pharmaceutical composition of thepresent invention comprising the at least one polymer conjugate alsocomprises a further agent as defined above. The pharmaceuticalcomposition of the present invention may be used for diagnostic or fortherapeutic applications.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's conditions.Administration may be achieved in a single dose or repeated doses atintervals. Dosage amount and interval may be adjusted individually inorder to provide the therapeutical effect which results in ameliorationof symptoms or a prolongation of the survival in a patient. The actualamount of composition administered will, of course, be dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgement of theprescribing physician. A suitable daily dosage will be between 0.001 to10 mg/kg, particularly 0.1 to 5 mg/kg.

The administration may be carried out by known methods, e.g. byinjection, in particular by intravenous, intramuscular, transmucosal,subcutaneous or intraperitoneal injection and/or by oral, topical,nasal, inhalation, aerosol and/or rectal application, etc. Theadministration may be local or systemic.

In addition, the polymer conjugates of the Box-A of HMGB1 moiety objectof this invention can be reversibly immobilized and/or adsorbed on thesurface and/or inside medical devices or drug release/vehicling systems(microspheres). Medical devices and microspheres can be reversiblyloaded with the polymer conjugates of this invention, through theirbinding, impregnation and/or adsorption on the surface of the medicaldevice or of the microsphere or on a layer that coats its surface. Whenthe medical device or the microsphere come into contact with biologicalfluids, the reversibly immobilized polymer conjugate is released.Therefore, the medical device and the microsphere act as drug-releasingtools that elute the molecule object of this invention in such a waythat their release kinetics can be controlled, ensuring controlled orsustained release, as required by the treatment. The methods forcoating/impregnating the medical devices and loading microspheres arewell known by experts in these technologies.

Thus, a further aspect of this invention is the use of the polymerconjugates of Box-A of HMGB1, wherein conjugated molecules arereversibly immobilized on the surface of medical devices or ofmicrospheres or are adsorbed within them. These medical instruments arepreferably surgical tools, implants, catheters or stents, for examplestents for angioplasty and, in particular, medicated drug-elutingstents.

Another aspect of the invention concerns a medical device reversiblycoated with at least one polymer conjugate of the invention. Such adevice can be selected from surgical instruments, implants, catheters orstents. Such a device may be useful for angioplasty.

The invention is further illustrated by the following figures:

FIG. 1a displays the amino acid sequence of the native Human HMGB1 Box-Amade of 84 amino acid residues (SEQ ID NO:1).

FIG. 1 b shows the type of replacing amino acids on the respectivetarget positions selected to generate the polypeptide variant of thefull-length human HMGB1 Box-A. Further, the specific amino acidsequences of the generated polypeptide variant are displayed in SEQ IDNOs:2 to 116.

FIG. 2a displays the amino acid sequence of the biologically activefragment of Human HMGB1 Box-A made of 77 amino acid residues (SEQ IDNO:117).

FIG. 2b shows the type of replacing amino acids on the respective targetpositions selected to generate the polypeptide variant of thebiologically active fragment of Human HMGB1 Box-A made of 77 amino acidresidues. Further the specific amino acid sequences of the generatedpolypeptide variant are displayed in SEQ ID NOs: 118 to 222.

FIG. 3a displays the amino acid sequence of the biologically activefragment of Human HMGB1 Box-A made of 54 amino acid residues (SEQ IDNO:223).

FIG. 3b shows the type of replacing amino acids on the respective targetpositions selected to generate the polypeptide variant of thebiologically active fragment of Human HMGB1 Box-A made of 54 amino acidresidues. Further, the specific amino acid sequences of the generatedpolypeptide variant are displayed in SEQ ID NOs: 224 to 300.

FIG. 4a displays the amino acid sequence of the native Anopheles gambiaHMGB1 Box-A made of 84 amino acid residues (SEQ ID NO:301).

FIG. 4b shows the type of replacing amino acids on the respective targetpositions selected to generate the polypeptide variant of thefull-length Anopheles gambia HMGB1 Box-A. Further, the specific aminoacid sequences of the generated polypeptide variant are displayed in SEQID NOs: 302 to 418.

FIG. 5a displays the amino acid sequence of the biologically activefragment of Anopheles gambia HMGB1 Box-A made of 77 amino acid residues(SEQ ID NO:419).

FIG. 5b shows the type of replacing amino acids on the respective targetpositions selected to generate the polypeptide variant of thebiologically active fragment of Anopheles gambia HMGB1 Box-A made of 77amino acid residues. Further the specific amino acid sequences of thegenerated polypeptide variant are displayed in SEQ ID NOs: 420 to 528.

FIG. 6a displays the amino acid sequence of the biologically activefragment of Anopheles gambia HMGB1 Box-A made of 54 amino acid residues(SEQ ID NO:529).

FIG. 6b shows the type of replacing amino acids on the respective targetpositions selected to generate the polypeptide variant of thebiologically active fragment of Anopheles gambia HMGB1 Box-A made of 54amino acid residues. Further, the specific amino acid sequences of thegenerated polypeptide variant are displayed in SEQ ID NOs: 530 to 610.

FIGS. 7A to 7I and Tables 7.1 to 7.9 show the results of the chemotaxisassay described of Example 1 performed on the HMGB1 Box-A polypeptidevariants of SEQ ID NO:2 to SEQ ID NO:116 used as HMGB1 Box-A moiety ofthe polymer conjugates of the present invention. In each figure theactivity of a set of polypeptide variants in the inhibition of HMGB1induced NIH/3T3 cell migration is tested compared to the activity ofhuman wild-type HMGB1 Box-A full-length fragment of SEQ ID NO:1. Eachfigure shows a table reporting the statistical analysis numerical dataand a column bar graph showing the chemotaxis assay results.

FIG. 7A and Table 7.1 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID NO:2 to 15 (identified in the Table andFigure with the code CT501, CT568, CT569, CT570, CT571, CT502, CT572,CT503, CT573, CT504, CT574, CT575, CT576 and CT505, respectively).

TABLE 7.1 MEAN Std (cells/filter) deviation SEM w/o FBS 2204 1.1190.3956 HMGB1 1 nM 51.13 2 702 09552  HMGB1 1 nM + CT500 1 nM 21 71 1 80306376 HMGB1 1 nM + CT501 1 nM 19.94 1 400 0.4950 HMGB1 1 nM + CT568 1 nM29.19 2506 0 8861 HMGB1 1 nM + CT569 1 nM 28.06 3 812 1 348  HMGB1 1nM + CT570 1 nM 30.00 4.559 1.612  HMGB1 1 nM + CT571 1 nM 3594 2528 08936 HMGB1 1 nM + CT502 1 nM 25 31 3 218 1138  HMGB1 1 nM + CT572 1 nM26.63 2.489 0.8801 HMGB1 1 nM + CT503 1 nM 18.75 3012 1 065  HMGB1 1nM + CT573 1 nM 26 31 4383 1 550  HMGB1 1 nM + CT504 1 nM 26.00 4.1491.467  HMGB1 1 nM + CT574 1 nM 31 19 2.789 09862 HMGB1 1 nM + CT575 1 nM2913 3824 1352  HMGB1 1 nM + CT576 1 nM 30.19 2404 0.8501 HMGB1 1 nM +CT505 1 nM 18 13 2900 1025 

FIG. 7B and Table 7.2 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID NOs:16-23 and 25-29 (identified in theTable and Figure with the code CT577, CT578, CT506, CT579, CT580, CT581,CT507, CT582, CT584, CT508, CT509, CT510 and CT585, respectively).

TABLE 7.2 MEAN Std (cells/filter) deviation SEM w/o FBS 20.25 1.0350.3660 HMGB1 1 nM 64.42 8.556 3.025 HMGB1 1 nM + CT500 1 nM 23.33 3.5051.239 HMGB1 1 nM + CT577 1 nM 34.75 2.171 0.7678 HMGB1 1 nM + CT578 1 nM29.56 3.396 1.201 HMGB1 1 nM + CT506 1 nM 25.31 3.936 1.392 HMGB1 1 nM +CT579 1 nM 51.31 4.140 1.464 HMGB1 1 nM + CT580 1 nM 30.44 3.469 1.226HMGB1 1 nM + CT581 1 nM 30.44 3.469 1.226 HMGB1 1 nM + CT507 1 nM 24.814.183 1.479 HMGB1 1 nM + CT582 1 nM 38.22 5.205 1.840 HMGB1 1 nM + CT5841 nM 30.56 2.796 0.9885 HMGB1 1 nM + CT508 1 nM 25.63 2.838 1.003 HMGB11 nM + CT509 1 nM 28.88 1.827 0.6461 HMGB1 1 nM + CT510 1 nM 25.50 5.2851.868 HMGB1 1 nM + CT585 1 nM 40.63 4.719 1.668

FIG. 7C and Table 7.3 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:30-35 and 37-43 (identified in theTable and Figure with the code CT511, CT512, CT513, CT514, CT586, CT515,CT516, CT517, CT518, CT519, CT520, CT521 and CT522, respectively).

TABLE 7.3 MEAN Std (cells/filter) deviation SEM w/o FBS 20.38 2.2850.8078 HMGB1 1 nM 72.54 5.188 1.834 HMGB1 1 nM + CT500 0.5 nM 33.312.375 0.8395 HMGB1 1 nM + CT511 0.5 nM 26.31 5.669 2.004 HMGB1 1 nM +CT512 0.5 nM 26.56 2.872 1.015 HMGB1 1 nM + CT513 0.5 nM 25.93 1.5120.5714 HMGB1 1 nM + CT514 0.5 nM 35.29 2.233 0.8441 HMGB1 1 nM + CT5860.5 nM 60.06 5.179 1.831 HMGB1 1 nM + CT515 0.5 nM 24.56 3.959 1.400HMGB1 1 nM + CT516 0.5 nM 29.09 29.49 1.043 HMGB1 1 nM + CT517 0.5 nM27.25 3.229 1.142 HMGB1 1 nM + CT518 0.5 nM 29.25 2.632 0.9306 HMGB1 1nM + CT519 0.5 nM 26.81 3.712 1.313 HMGB1 1 nM + CT520 0.5 nM 27.313.047 1.077 HMGB1 1 nM + CT521 0.5 nM 29.13 2.888 1.021 HMGB1 1 nM +CT522 0.5 nM 25.69 3.391 1.199

FIG. 7D and Table 7.4 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NOs:44-57(identified in the Table and Figure with the code CT523, CT524, CT525,CT526, CT527, CT528, CT588, CT529, CT530, CT589, CT590, CT531, CT591 andCT532, respectively).

TABLE 7.4 MEAN Std (cells/filter) deviation SEM w/o FBS 20.27 2.2500.7955 HMGB1 1 nM 66.58 6.732 2.380 HMGB1 1 nM + CT500 0.5 nM 36.503.045 1.076 HMGB1 1 nM + CT523 0.5 nM 34.06 3.849 1.361 HMGB1 1 nM +CT524 0.5 nM 39.57 6.380 2.411 HMGB1 1 nM + CT525 0.5 nM 41.06 4.2291.495 HMGB1 1 nM + CT526 0.5 nM 34.13 4.764 1.684 HMGB1 1 nM + CT527 0.5nM 29.88 3.182 1.125 HMGB1 1 nM + CT528 0.5 nM 41.50 2.878 1.018 HMGB1 1nM + CT588 0.5 nM 60.13 5.848 2.067 HMGB1 1 nM + CT529 0.5 nM 30.133.357 1.187 HMGB1 1 nM + CT589 0.5 nM 35.63 2.504 0.8851 HMGB1 1 nM +CT590 0.5 nM 43.88 3.227 1.141 HMGB1 1 nM + CT531 0.5 nM 47.00 2.5350.8984 HMGB1 1 nM + CT591 0.5 nM 35.25 8.045 2.844 HMGB1 1 nM + CT5320.5 nM 43.56 3.287 1.155 HMGB1 1 nM + CT500 0.5 nM 26.50 3.094 1.094

FIG. 7E and Table 7.5 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:58-67 and 69-71 (identified in theTable and Figure with the code CT592, CT533, CT593, CT534, CT535, CT536,CT537, CT594, CT538, CT539, CT540, CT541 and CT542, respectively).

TABLE 7.5 MEAN Std (cells/filter) deviation SEM w/o FBS 18.48 1.6940.5988 HMGB1 1 nM 76.81 5.000 1.768 HMGB1 1 nM + CT500 0.5 nM 35.900.738 0.2790 HMGB1 1 nM + CT592 0.5 nM 43.00 4.041 1.528 HMGB1 1 nM +CT533 0.5 nM 35.88 4.883 1.726 HMGB1 1 nM + CT593 0.5 nM 47.14 1.5740.5948 HMGB1 1 nM + CT534 0.5 nM 34.00 3.742 1.323 HMGB1 1 nM + CT5350.5 nM 33.21 3.534 1.336 HMGB1 1 nM + CT536 0.5 nM 28.00 1.558 0.5510HMGB1 1 nM + CT537 0.5 nM 28.88 2.925 1.034 HMGB1 1 nM + CT594 0.5 nM45.31 3.391 1.199 HMGB1 1 nM + CT538 0.5 nM 31.93 3.421 1.293 HMGB1 1nM + CT539 0.5 nM 34.41 3.265 1.154 HMGB1 1 nM + CT540 0.5 nM 29.811.850 0.6542 HMGB1 1 nM + CT541 0.5 nM 27.44 2.195 0.7760 HMGB1 1 nM +CT542 0.5 nM 32.19 5.411 1.913

FIG. 7F and Table 7.6 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:72-85 (identified in the Table andFigure with the code CT596, CT597, CT598, CT599, CT600, CT601, CT602,CT603, CT543, CT544, CT545, CT546, CT547 and CT604, respectively).

TABLE 7.6 MEAN Std (cells/filter) deviation SEM w/o FBS 15.25 2.2310.7887 HMGB1 1 nM 90.04 5.400 1.909 HMGB1 1 nM + CT500 0.5 nM 34.041.713 0.6057 HMGB1 1 nM + CT596 0.5 nM 55.56 3.479 1.230 HMGB1 1 nM +CT597 0.5 nM 92.79 11.77 4.449 HMGB1 1 nM + CT598 0.5 nM 64.38 4.4461.572 HMGB1 1 nM + CT599 0.5 nM 58.81 6.681 2.362 HMGB1 1 nM + CT600 0.5nM 95.86 7.063 2.670 HMGB1 1 nM + CT601 0.5 nM 67.44 7.302 2.582 HMGB1 1nM + CT602 0.5 nM 49.63 2.532 0.8952 HMGB1 1 nM + CT603 0.5 nM 41.563.923 1.387 HMGB1 1 nM + CT543 0.5 nM 41.44 2.884 1.020 HMGB1 1 nM +CT544 0.5 nM 30.63 1.620 0.5728 HMGB1 1 nM + CT545 0.5 nM 40.13 3.5831.267 HMGB1 1 nM + CT546 0.5 nM 34.88 4.051 1.432 HMGB1 1 nM + CT547 0.5nM 41.64 4.661 1.762 HMGB1 1 nM + CT604 0.5 nM 61.85 5.330 1.885

FIG. 7G and Table 7.7 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID NOs:86-99 (identified in the Table andFigure with the code CT548, CT549, CT605, CT606, CT607, CT608, CT609,CT610, CT550, CT551, CT611, CT552, CT553 and CT554, respectively).

TABLE 7.7 MEAN Std (cells/filter) deviation SEM w/o FBS 24.33 1.8690.6607 HMGB1 1 nM 90.58 2.888 1.021 HMGB1 1 nM + CT500 0.5 nM 44.334.673 1.652 HMGB1 1 nM + CT546 0.5 nM 45.38 3.068 1.085 HMGB1 1 nM +CT549 0.5 nM 44.56 4.362 1.542 HMGB1 1 nM + CT605 0.5 nM 84.63 5.6431.995 HMGB1 1 nM + CT606 0.5 nM 83.19 5.182 1.832 HMGB1 1 nM + CT607 0.5nM 68.00 4.132 1.461 HMGB1 1 nM + CT608 0.5 nM 89.50 6.503 2.299 HMGB1 1nM + CT609 0.5 nM 89.56 3.110 1.100 HMGB1 1 nM + CT610 0.5 nM 82.195.398 1.908 HMGB1 1 nM + CT550 0.5 nM 28.06 3.479 1.230 HMGB1 1 nM +CT551 0.5 nM 37.50 4.862 1.719 HMGB1 1 nM + CT611 0.5 nM 55.88 4.0601.435 HMGB1 1 nM + CT552 0.5 nM 42.94 3.510 1.241 HMGB1 1 nM + CT553 0.5nM 40.25 4.097 1.449 HMGB1 1 nM + CT554 0.5 nM 43.69 2.235 0.7902

FIG. 7H and Table 7.8 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:100-113 (identified in the Table andFigure with the code CT555, CT556, CT557, CT558, CT559, CT612, CT560,CT561, CT613, CT562, CT563, CT564, CT565 and CT566, respectively).

TABLE 7.8 MEAN Std (cells/filter) deviation SEM w/o FBS 17.10 2.4280.859 HMGB1 1 nM 75.90 3.613 1.277 HMGB1 1 nM + CT500 0.5 nM 33.33 2.6430.934 HMGB1 1 nM + CT555 0.5 nM 26.13 2.151 0.760 HMGB1 1 nM + CT556 0.5nM 30.13 2.774 0.981 HMGB1 1 nM + CT557 0.5 nM 33.63 5.397 1.908 HMGB1 1nM + CT558 0.5 nM 25.00 3.064 1.573 HMGB1 1 nM + CT559 0.5 nM 26.944.448 1.083 HMGB1 1 nM + CT612 0.5 nM 65.13 4.948 1.749 HMGB1 1 nM +CT560 0.5 nM 27.50 2.891 1.022 HMGB1 1 nM + CT561 0.5 nM 27.13 2.9731.051 HMGB1 1 nM + CT613 0.5 nM 43.06 2.337 0.826 HMGB1 1 nM + CT562 0.5nM 28.19 1.602 0.567 HMGB1 1 nM + CT563 0.5 nM 27.75 3.381 1.195 HMGB1 1nM + CT564 0.5 nM 23.38 1.747 0.618 HMGB1 1 nM + CT565 0.5 nM 29.002.121 0.750 HMGB1 1 nM + CT566 0.5 nM 27.75 2.220 0.785

FIG. 7I and Table 7.9 show the bar graph and statistical data ofchemotaxis migration assay results in the inhibition of HMGB1-inducedNIH/3T3 cells by human HMGB1 Box-A wild type of SEQ ID NO:1 (CT500) andpolypeptide variants of SEQ ID Nos:114-116 (identified in the Table andFigure with the code CT567, CT614 and CT615, respectively).

TABLE 7.9 MEAN Std (cells/filter) deviation SEM w/o FBS 14.04 1.3150.4648 HMGB1 1 nM 62.96 1.864 0.659 HMGB1 1 nM + CT500 0.5 nM 21.712.155 0.815 HMGB1 1 nM + CT567 0.5 nM 19.31 2.052 0.725 HMGB1 1 nM +CT614 0.5 nM 28.71 2.119 0.801 HMGB1 1 nM + CT615 0.5 nM 39.81 2.1540.761

FIG. 8 shows the image of the Tricine SDS-PAGE gel loaded with humanHMGB1 Box-A wild type of SEQ ID NO:1 (CT500) at different timepointsafter protease digestion of the protease resistance testing described inExample 2 The Box-A wild type protein tested for protease resistance isa His-tagged protein. After 5 minutes of digestion CT500 shows two majorbands, one corresponding to the original protein in the sample and thesecond corresponding to the 84-aminoacid protein without the N-termHis-tag (indicated on the figure with an arrow). The profile of thissecond band shows resistance to proteases for 30 minutes. Minor bandspresent on this and other gels of FIG. 9A to FIG. 9BO correspond toBox-A digested fragments.

FIG. 9A to FIG. 9BO show the image of the Tricine SDS-PAGE gel loadedwith the polypeptide variants of the HMGB1 Box-A moiety of the polymerconjugates of the present invention at different timepoints afterprotease digestion of the protease resistance testing described inExample 2. Box-A polypeptide variants tested for protease resistance areHig-tagged proteins. After 5 minutes of digestion the SDS-PAGE gel imageof the polypeptide variants show two major bands, one corresponding tothe original protein variant in the sample and the second correspondingto the Box-A 84 amino acid protein variant without the N-term His-tag.

FIG. 9A shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:2 (CT501) at different timepoints after protease digestion.

FIG. 9B shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:7 (CT502) at different timepoints after protease digestion.

FIG. 9C shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:9 (CT503) at different timepoints after protease digestion.

FIG. 9D shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:11 (CT504) at different timepoints after protease digestion.

FIG. 9E shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:15 (CT505) at different timepoints after protease digestion.

FIG. 9F shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:18 (CT506) at different timepoints after protease digestion.

FIG. 9G Tricine SDS-PAGE of the polypeptide variant of SEQ ID NO:22(CT507) at different timepoints after protease digestion.

FIG. 9H shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:26 (CT508) at different timepoints after protease digestion.

FIG. 9I shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:27 (CT509) at different timepoints after protease digestion.

FIG. 9J shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:28 (CT510) at different timepoints after protease digestion.

FIG. 9K shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:30 (CT511) at different timepoints after protease digestion.

FIG. 9L shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:31 (CT512) at different timepoints after protease digestion.

FIG. 9M shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:32 (CT513) at different timepoints after protease digestion.

FIG. 9N shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:33 (CT514) at different timepoints after protease digestion.

FIG. 9O shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:35 (CT515) at different timepoints after protease digestion.

FIG. 9P shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:37 (CT516) at different timepoints after protease digestion.

FIG. 9Q shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:38 (CT517) at different timepoints after protease digestion.

FIG. 9R shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:39 (CT518) at different timepoints after protease digestion.

FIG. 9S shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:40 (CT519) at different timepoints after protease digestion.

FIG. 9T shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:41 (CT520) at different timepoints after protease digestion.

FIG. 9U shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:42 (CT521) at different timepoints after protease digestion.

FIG. 9V shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:43 (CT522) at different timepoints after protease digestion.

FIG. 9W shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:44 (CT523) at different timepoints after protease digestion.

FIG. 9X shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:45 (CT524) at different timepoints after protease digestion.

FIG. 9Y shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:46 (CT525) at different timepoints after protease digestion.

FIG. 9Z shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:47 (CT526) at different timepoints after protease digestion.

FIG. 9AA shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:48 (CT527) at different timepoints after protease digestion.

FIG. 9AB shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:49 (CT528) at different timepoints after protease digestion.

FIG. 9AC shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:51 (CT529) at different timepoints after protease digestion.

FIG. 9AD shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:52 (CT530) at different timepoints after protease digestion.

FIG. 9AE shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:55 (CT531) at different timepoints after protease digestion.

FIG. 9AF shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:57 (CT532) at different timepoints after protease digestion.

FIG. 9AG shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:59 (CT533) at different timepoints after protease digestion.

FIG. 9AH shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:61 (CT534) at different timepoints after protease digestion.

FIG. 9AI shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:62 (CT535) at different timepoints after protease digestion.

FIG. 9AJ shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:63 (CT536) at different timepoints after protease digestion.

FIG. 9AK shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:64 (CT537) at different timepoints after protease digestion.

FIG. 9AL shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:66 (CT538) at different timepoints after protease digestion.

FIG. 9AM shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:67 (CT539) at different timepoints after protease digestion.

FIG. 9AN shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:69 (CT540) at different timepoints after protease digestion.

FIG. 9AO shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:70 (CT541) at different timepoints after protease digestion.

FIG. 9AP shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:71 (CT542) at different timepoints after protease digestion.

FIG. 9AQ shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:80 (CT543) at different timepoints after protease digestion.

FIG. 9AR shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:81 (CT544) at different timepoints after protease digestion.

FIG. 9AS shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:82 (CT545) at different timepoints after protease digestion.

FIG. 9AT shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:83 (CT546) at different timepoints after protease digestion.

FIG. 9AU shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:84 (CT547) at different timepoints after protease digestion.

FIG. 9AV shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:86 (CT548) at different timepoints after protease digestion.

FIG. 9AW shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:87 (CT549) at different timepoints after protease digestion.

FIG. 9AX shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:94 (CT550) at different timepoints after protease digestion.

FIG. 9AY shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:95 (CT551) at different timepoints after protease digestion.

FIG. 9AZ shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:97 (CT552) at different timepoints after protease digestion.

FIG. 9BA shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:98 (CT553) at different timepoints after protease digestion.

FIG. 9BB shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:99 (CT554) at different timepoints after protease digestion.

FIG. 9BC shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:100 (CT555) at different timepoints after protease digestion.

FIG. 9BD shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:101 (CT556) at different timepoints after protease digestion.

FIG. 9BE shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:102 (CT557) at different timepoints after protease digestion.

FIG. 9BF shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:103 (CT558) at different timepoints after protease digestion.

FIG. 9BG shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:104 (CT559) at different timepoints after protease digestion.

FIG. 9BH shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:106 (CT560) at different timepoints after protease digestion.

FIG. 9BI shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:107 (CT561) at different timepoints after protease digestion.

FIG. 9BJ shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:109 (CT562) at different timepoints after protease digestion.

FIG. 9BK shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:110 (CT563) at different timepoints after protease digestion.

FIG. 9BL shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:111 (CT564) at different timepoints after protease digestion.

FIG. 9BM shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:112 (CT565) at different timepoints after protease digestion.

FIG. 9BN shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:113 (CT566) at different timepoints after protease digestion.

FIG. 9BO shows the Tricine SDS-PAGE of the polypeptide variant of SEQ IDNO:19 (CT567) at different timepoints after protease digestion.

FIG. 10 shows a table in which the results of the Tricine SDS-PAGE aresummarized. A cross indicates the presence on the gel of the bandcorresponding to the 84 amino acid long protein fragment of the HMGB1Box-A wild-type or of the HMGB1 Box-A polypeptide variant.

FIG. 11 shows the mean plasma concentration/time after a singlesubcutaneous administration of Box A wild type (WT) and Box A variantnumber 64 (64) at a dosage of 1 mg/kg. Data representation: Mean±SEM.

FIG. 12 shows the mean plasma concentration/time after a singlesubcutaneous administration of Box A wild type (WT) and Box A variantnumber 64 (64) at a dosage of: 1 mg/kg and of PEGylated Box A wild type(PEG-WT) and PEGylated Box A variant number 64 (PEG-64) at a dosage of 5mg/kg. Data representation: Mean±SEM.

EXAMPLES 1. In Vitro Activity Testing: NIH/373 Cell Migration Assay

The purpose of the present study was to evaluate the activity of each ofthe HMGB1 Box-A polypeptide variants as defined in SEQ ID NOs:2-116 andto compare their activity to that of human wild type HMGB1 Box-Afull-length fragment of SEQ ID NO:1 in order to select all the variantswith similar or better activity than wild type.

HMGB1 Box-A activity is evaluated in vitro as inhibition ofHMGB1-induced NIH/3T3 cells migration.

1.1 Materials

-   -   HMGB1 Box-A wild type and variants (Nautilus Biotech)    -   NIH/3T3 cells (ATCC n. CRL-1658)    -   D-MEM medium (GIBCO; cat. n. 31966-021)    -   Foetal Bovine Serum (GIBCO; cat. n. 10270-106)    -   Penicillin-Streptomycin 10,000 U/ml (GIBCO; cat. n. 15140-122)    -   L-Glutamine 200 mM (GIBCO; cat. n. 25030-024)    -   TrypLE Select (GIBCO; cat. n. 12563-011)    -   Phosphate Buffered Saline (0.138 M NaCl, 0.0027 M KCl, 0.01 M        phosphate, pH 7.4)    -   PVP free filters (8 μm pore size; 13 mm total diameter) (Neuro        Probe; cat. n. PFA8)    -   Human fibronectin (Roche; cat. n. 1080938)    -   Blind Well Chemotaxis Chambers (Neuro Probe; cat. n. BW25)    -   GIEMSA Stain Modified (Sigma; cat. n. GS1L)        1.2 Filters Preparation

Polycarbonate membranes PVP free filters (8 μm pore size, 13 mm totaldiameter) are prepared about one hour before performing the experimentby coating them with 30 μl/filter of a solution 50 μg/ml of fibronectindispensed on the opaque side of the filter. The stock fibronectinsolution is prepared by diluting the lyophilized fibronectin in ddH₂O toa final concentration of 1 mg/ml and by keeping the solution about 1hour at 37° C. for complete dissolution. This stock solution can bestored at −20° C.

The filters are then left to dry under the laminar flux of the hood(about one hour).

1.3 Cells Preparation

NIH/3T3 cells are seeded the day before the experiment (approximately22-24 hours before performing the experiment) 10⁶ cells/plate.

When the filters are ready to use, the cells are detached with Trypsin,counted and resuspended 10⁶ cells/ml in serum free culture medium.

1.4 Chemotaxis Assay

In each chemotaxis experiment 14 different polypeptide variants of thehuman HMGB1 Box-A full-length fragment of SEQ ID NO:1 are tested.

Growth cell medium without serum addition (w/o FBS) is used as negativecontrol representing spontaneous migration.

1 nM HMGB1 is used as positive control. HMGB1 Box-A wild type or thetested polypeptide variants 0.5/1 nM are added to 1 nM HMGB1 to inhibitHMGB1-induced NIH/3T3 cell migration.

Negative control (w/o FBS) and positive control (1 nM HMGB1) are testedin triplicate in each experiment.

HMGB1 Box-A wild type (SEQ ID NO:1) activity in inhibiting HMGB1-inducedcell migration is tested in triplicate in each experiment.

Each of the HMGB1 Box-A polypeptide variants (SEQ ID NOs:2 to 116) istested in duplicate.

Blind Well Chemotaxis Chambers are used. The clean, dry lower well ofeach chamber is filled with 50 μl of DMEM without FBS added with theappropriate chemotactic agent and inhibitors. A slight positive meniscusshould form when the well is filled; this helps prevent air bubbles frombeing trapped when the filter is applied. With small forceps the filteris placed over the filled well (fibronectin treated side up), beingcareful not to trap air bubbles and not to touch the filter withfingers. The filter retainer is screwed in by hand. Cell suspension(50000 cells/50 μl) is pipetted into the upper well and 150 μl of serumfree medium are added to fill the upper well of the chamber. The filledchamber is incubated for 3 hours (37° C., 5% CO₂) to allow cellmigration. After incubation the fluid is removed from the filter. Theretainer is unscrewed and immersed in cool distilled water. The filteris lifted out with forceps, placed on a clean surface (solid paraffin)(migrated cells side up) and fixed with a needle (placed on the borderarea).

1.5 GIEMSA Staining of Migrated Cells

The filters are fixed with ethanol once and then washed three timesunder running water. A working solution of GIEMSA Stain Modified diluted1:10 in ddH₂O is prepared just before use. After washing of the filters,the staining is added and left to incubate for 20 minutes. Washing ofthe staining is performed under running water. The filters are thenplaced on slides with the migrated cells side down, and the non-migratedcells side is gently wiped off with a wet cotton swab (wipe twice, usingtwo swabs or both ends of a double-tipped swab) being careful not tomove the filter. After cleaning, a cover slide is placed on the filterand cells are counted under a microscope at 40× in 10 randomfields/filter.

1.6 Data Representation and Statistical Analysis

The results of the NIH/3T3 migration assay performed are reported in thetables and bar graphs shown in Figure and Table 7.1 to Figure and Table7.9.

Data are represented in bar columns as MEAN±95% CI.

One-way ANOVA followed by Dunnett's post test (control column data: 1 nMHMGB1 sample+HMGB1 Box-A WT sample) is the statistical analysisperformed.

When evaluating the results data, HMGB1 Box-A variants data having apost test p value<0.05 are considered significantly different from HMGB1Box-A wild type. If the mean of the Box-A polypeptide variant is higherthan that of Box-A wild type the column is coloured in red in the graphof the experiment shown in FIGS. 7.1 to 7.9. Those red columns representHMGB1 Box-A polypeptide variants showing less activity than wild type ininhibiting HMGB1-induced cell migration.

If the mean of the polypeptide variant results lower than that of wildtype Box-A then the column is coloured in light blue in the graph of theexperiment shown in FIGS. 7.1 to 7.9. Those variants represent HMGB1Box-A variants showing higher activity than HMGB1 Box-A wild type ininhibiting HMGB1-induced cell migration.

HMGB1 Box-A variants data having a post test p value>0.05 are considerednot significantly different from HMGB1 Box-A wild type. The bar columnof those variants are coloured in green. Those variants represent HMGB1Box-A variants showing the same activity of wild type in inhibitingHMGB1-induced cell migration.

1.7 Results

The activity of polypeptide variants of the human HMGB1 high affinitybinding domain Box-A of SEQ ID NOs:2 to 116 was evaluated in comparisonto human HMGB1 Box-A wild-type of SEQ ID NO:1 as inhibition ofHMGB1-induced cell migration, in order to determine the preferredpolypeptide variants useful as HMGB1 Box-A moiety of the preferredpolymer conjugate of the present invention.

The chemotaxis assays results revealed (FIGS. 7.1 to 7.9) that for 26polypeptide variants the mutation according to the present inventioncould lead to a higher activity in cell migration inhibition incomparison with the activity of the wild-type human HMGB1 Box-A. Inparticular, a higher activity in cell migration inhibition was shown forthe polypeptide variants of SEQ ID NOs: 30-32, 35, 38, 40-41, 43, 48,51, 57, 63-64, 69, 70, 94, 95, 100, 103-104, 106-107, 109-111 and 113.

Moreover, the chemotaxis assays results revealed (FIGS. 7.1 to 7.9) that41 polypeptide variants showed no changes in their activity ininhibiting HMGB1-induced cell migration compared to the activity ofBox-A wild-type polypeptide. In particular, this is the case for thepolypeptide variants of SEQ ID NOs: 2, 7, 9, 11, 15, 18, 22, 26-28, 33,37, 39, 42, 44-47, 49, 52, 55, 59, 61, 62, 66-67, 71, 80-84, 86-87,97-99, 101-102, 112 and 114.

All these Box-A polypeptide variants which exhibit a similar or a higheractivity than the Box-A wild-type were tested for in vitro proteaseresistance, in order to choose the most resistant ones that are at leastas active as Box-A wild-type (see protease resistance test in Example2).

2. In Vitro Protease Resistance Testing

The purpose of the present study was to evaluate the in vitro proteaseresistance of to HMGB1 Box-A variants shown in Example 1 and to compareit to that of wild type HMGB1 Box-A of SEQ ID NO:1 in order to identifythe variants with improved protease resistance with respect to wild typepolypeptide.

2.1 Materials

-   -   HMGB1 His-tagged Box-A wild type and selected variants (Nautilus        biotech)    -   Trypsin (Sigma; cat. n. T8658; lot. n. 045K5113)    -   α-chymotrypsin (Sigma; cat. n. C6423; lot. n. 109H74858),    -   Endoproteinase Asp-N (Sigma; cat. n. P3303; lot. n. 046K1049)    -   Endoproteinase Glu-C (Sigma; cat. n. P6181; lot. n. 075K5100)    -   Complete, Mini EDTA-free protease inhibitor cocktail (Roche;        cat. n. 11836170 001)    -   Trizma base (Sigma; cat. n. T6066)    -   Acrylamide/bis solution 40% in water (Sigma; cat. n. 01709)    -   SDS (Sigma; cat. n. 71729)    -   Glycerol 99% (Sigma; cat. n. G9012)    -   Temed (Sigma; cat. n. 87689)    -   APS (Sigma; cat. n. A 3678)    -   Polypeptide SDS-PAGE Molecular Weight Standards (Bio-Rad;        cat. n. 161-0326)    -   Premixed 10× Tris/tricine/SDS Buffer (Bio-Rad; cat. n. 161-0744)    -   β-Mercaptoethanol (Sigma; cat. n. M7154)    -   Methanol (VWR; cat. n. 20864.320)    -   Acetic acid (VWR; cat. n. 20104.323)    -   Brilliant Blue R (Sigma; cat. n. B0149)    -   Bromophenol Blue (Sigma; cat. n. B0126)    -   Hydrochloric acid (Merck; cat. n. 1.00319.2511)    -   3× sample loading buffer for Tricine gels (composition: 150 mM        Tris-HCl, pH 6.8; 12% SDS; 36% glycerol; 6% β-Mercaptoethanol;        0.04% of bromophenol blue)        2.2. Protease Mixture Preparation

A mixture of proteases containing trypsine, α-chymotrypsine,endoproteinase Asp-N and endoproteinase Glu-C is used.

Table 1 reports specificity of each of the proteases used in this study.

TABLE 1 protease specificity. Protease Specificity Trypsin C-term of K,R (not if P at C-term of cutting site; slower digestion if acidicresidue on either side of cutting site) α-chymotrypsin C-term of T, P,W, L (secondary hydrolysis: C-term of M, I, S, T, V, H, G, A)Endoproteinase Asp-N N-term of D, C Endoproteinase Glu C-term of E, D(not if P is at C-term of cutting site)

Each lyophilized protease is dissolved according to manufacturerrecommendations to obtain a stock solution that is aliquoted and storedat −80° C.

100 μg of trypsin are dissolved in 100 μl of dH₂O to obtain a 1 μg/μlstock solution. 25 μg of α-chymotrypsine are dissolved in 50 μl of asolution 1 mM HCl, 2 mM CaCl₂ to obtain a 0.5 μg/μl stock solution. 2 μgof endoproteinase Asp-N are dissolved in 50 μl of dH₂O to obtain a 0.04μg/μl stock solution. 25 μg of endoproteinase Glu-C are dissolved in 50μl of dH₂O to obtain a 0.5 μg/μl stock solution.

Before performing the experiment one aliquot of each protease stocksolution is left to thaw on ice.

Trypsin and endoproteinase Glu-C stock aliquots are diluted in dH₂O toobtain a final working solution of 0.1 μg/μl. α-chymotrypsine stockaliquot is diluted in a solution 1 mM HCl, 2 mM CaCl₂ to obtain a final0.1 μg/μl working solution. Endoproteinase Asp-N aliquot is used withoutdilution.

Just before performing the experiment a mixture of proteases containing1% (in weight/weight of total Box-A contained in the sample) of eachprotease is freshly prepared and immediately added to HMGB1 Box-A to bedigested.

2.3. HMGB1 Box-A Wild Type and Variants Protease Digestion

18 μg total of each HMGB1 Box-A (wild type or variants) are digested ineach experiment.

HMGB1 Box-A to be tested is left to thaw on ice and the volumecorresponding to 18 μg is taken. The volume of this solution is thenbrought with dH₂O to a final volume of 90 μl in order to obtain the samefinal volume for each HMGB1 Box-A to be tested.

10 μl of this solution (corresponding to 2 μg of HMGB1 Box-A) are takenbefore adding the protease mixture. This sample corresponds to “time 0”not digested sample.

The remaining sample (16 μg of HMGB1 Box-A) is added with 8.8 μl(corresponding to 0.16 μg of each protease of the freshly preparedmixture; see 2.2) of protease mixture for digestion.

Protease digestion is performed at 25° C. and a volume corresponding to2 μg of HMGB1 Box-A (originally present in the mixture) is sampled atdefined time points. Digestion is stopped adding 4 μl of a solution ofcomplete Mini EDTA-free protease inhibitor cocktail (1 tablet dissolvedin 10 ml of dH₂O). Timepoints for sampling are: 0, 5 minutes, 15minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours and 4 hours.

Soon after protease inhibition, samples are added with the appropriateamount of sample loading buffer 3× and incubated at 95° C. for about 3minutes.

2.4. Tricine SDS-PAGE of Digested HMGB1 Box-A Wild Type and Variants

After protease digestion and samples preparation, timepoints samples ofeach HMGB1 Box-A are loaded on a Tricine SDS PAGE gel (see forreferences: Schägger and von Jagow, “Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa”, Anal. Biochem. 166, 368-379,1987).

5 μl of Polypeptide SDS-PAGE Molecular Weight Standards (Bio-Rad) areloaded for reference on each gel.

Each well of the gel is loaded with 10 μl of sample (volumecorresponding to 1 μg of HMGB1 Box-A before digestion).

Electrophoresis is performed at 30 V until the bromophenol blue hasentered the separating portion of the gel, then at 120 V (Mini Protean 3System; Bio-Rad) till the end of the run.

Gels are stained by soaking in a Coomassie Brilliant Blue R stainingsolution (0.1% w/v in 50% methanol, 10% acetic acid) for 1 hour anddestained overnight in destaining solution (30% methanol, 10% aceticacid).

Gel images are acquired with Gel Doc 2000 (Bio-Rad) imaging system.

2.5 Results

In the above reported assay conditions HGMB1 wild-type protein resistedapproximately 30 minutes to complete protease digestion. In FIG. 8 theband corresponding to the 84-amino acid full-length fragment of humanHMGB1 Box-A wild-type of SEQ ID NO:1 protein is visible until 30 minutesof protease digestion.

21 Box-A polypeptide variants tested showed an increased resistance toprotease (FIG. 10). In the reported assay conditions these variantsresist from 1 hour to 2 hours to protease digestion. The polypeptidevariants of SEQ ID NOs: 33, 35, 37, 38, 39, 42, 43, 44, 47, 48, 57, 62,69 and 104 showed a resistance of 1 hour to protease digestion. FIGS.9.14, 9.15, 9.16, 9.17, 9.18, 9.21, 9.22, 9.23, 9.26, 9.27, 9.32, 9.35,9.40 and 9.59 show a band corresponding to the not His-tagged protein of84 amino acids which is visible until 1 hour of protease digestion.

The polypeptide variants of SEQ ID NOs: 45, 49, 52, 55 and 67 showed aresistance of 1.5 hours to protease digestion. FIGS. 9.24, 9.28, 9.30,9.31 and 9.39 show a band corresponding to the not His-tagged protein of84 amino acids which is clearly visible 1 hour and a half after proteasedigestion. The polypeptide variants of SEQ ID NOs: 59 and 64 even show aresistance of up to 2 hours to protease digestion. FIGS. 9.33 and 9.37show a band of the not His-tagged protein of 84 amino acids which isclearly visible until 2 hours after protease digestion.

3. Pharmacokinetic Study of Box A (Wild Type and Variants) and PEGylatedBox A (Wild Type and Variants) after Single Subcutaneous Administrationin Mice

3.1. Aim of the Study

The purpose of the study was to evaluate and to compare thepharmacokinetic profile of Box A (wild type and variants) and PEGylatedBox A (wild type and variants) following a single subcutaneousadministration of the compounds in mice.

3.2. Materials and Methods

-   -   Test articles: Box A wild type and variants and the        corresponding PEGylated molecules. PEGylated molecules were        obtained reacting linear mPEG-aldehyde (40 kDa) with the        N-terminus of the respective Box A molecule by reductive        amination.        3.3 In Vivo Experiment    -   Animals: mice (Balb/c, males, 7-9 weeks old, supplied by Charles        River Laboratories Italia SpA, Calco one week before the        experiment) with an average body weight of 22.2-22.4 g at the        moment of the experiment.    -   Animal husbandry: the animals were housed in a ventilated        thermostatic container set to maintain temperature and relative        humidity at 22° C.±2° C. and 55±15% respectively, with 12 hours        light/dark cycle. Mice were housed up to 10 to a cage, in clear        polycarbonate cages (Techniplast, Buguggiate, Italy); drinking        water via water bottles and a commercially available laboratory        rodent diet (4RF21, Mucedola s.r.l., Settimo Milanese, Italy)        were supplied ad libitum.    -   Experimental groups: 4 (four test items), 20 animals/group,        randomly grouped.    -   Administered dose: Box A wild type and variants were        administered 1 mg/kg subcutaneously. PEGylated Box A wild type        and variants were administered 5 mg/kg subcutaneously. These        doses ensured equimolarity of test compounds.    -   Administration of the test items: test items were administered        subcutaneously by using an insulin syringe fitted with a 0.45×12        mm (26G×½″) needle at a volume of 0.25 mL/mouse (10 mL/kg body        weight).    -   Test article formulation: Phosphate Buffer Saline solution    -   Animal sacrifice and blood collection:        Blood samples were collected at the following time points after        treatment:    -   Box A wild type and variants: 5, 20 and 40 minutes, 1.5 and 2.5        hours after administration of the test compounds;    -   PEGylated Box A wild type and variants: 5, 40 minutes, 1.5, 5        and 10 hours after administration of the test compounds.        -   Different timepoints for blood sample collection between            test compounds were decided on the basis of longer expected            permanence of PEGylated molecules in the bloodstream.        -   At each sampling time, approximately 0.4 mL blood samples            were collected from the ventral aorta of each animal using            an insulin syringe, under deep ether anesthesia, and            transferred into polyethylene Eppendorf tubes containing 5            μL heparin (5000 Ul/mL) to prevent blood clotting. Blood            samples were kept in ice until centrifugation at 1400 g for            5 min. in a refrigerated centrifuge (2-4° C.). From each            tube plasma samples were then recovered, put in new            Eppendorf tubes and frozen at −80° C. until analysis.            3.4 Analytical Determination

Box A (wild type and variants) and PEGylated Box A (wild type andvariants) plasma concentrations were determined in mouse plasma by anELISA method.

Briefly, a coating solution was prepared by diluting a monoclonalantibody against the N-terminal of Box A to 10 ng/ml in 100 mMcarbonate-bicarbonate coating buffer. 100 μL were aliquoted to everywell of a Nunc Maxisorp ELISA plate, which was incubated overnight at 4°C. The plate was washed with PBS 0.05% Tween for 6 times and 300 μL of5% milk in PBS 1% Tween were added to each well to block the remainingbinding sites on the plate. The plate was incubated for 1 hour at roomtemperature at 300 rpm. Samples were diluted 1:20 in PBS 1% Tween.

The plate was washed 6-fold and 100 μL of standards and diluted sampleswere transferred to the designated wells of the coated plate andincubated at room temperature for 1 hour at 300 rpm. The plate waswashed 6-fold again prior to the addition of the secondary antibodyagainst the C-Terminal of Box A (1:200). After 1 hour of incubation and6 washes, the biotin-goat anti-rabbit conjugate 1:20000 solution wasadded to every well (100 μL/well). After 1 hour of incubation (at roomtemperature, 300 rpm) and six washes, the plate was incubated with 100μl/well of the streptavidin-HRP solution 1:100000 for 25 minutes at 300rpm. The plate was washed 6 times and 100 μl of pre-warmed TMB substratewere added to each well. The signal was developed at room temperature onthe bench top and, after 30 minutes, 100 μL/well of Stop Solution wereadded and the plate was immediately read at 450 nm.

3.5 Results

Mean plasma concentrations of Box A (wild type and variants) andPEGylated Box A (wild type and variants) were calculated for each of thepreviously described PK samples and the pharmacokinetic profiledetermined. The results are shown in FIGS. 11 and 12.

As example, here below are reported the PK profiles of Box A wild typeand variant n. 64 (M51I) and of PEGylated Box A and PEGylated Box Avariant number 64 (M51I). The results are reported as mean values±error(SEM) (4 mice for each time point, analysis in duplicate).

In the following table, the calculated AUC_(last) (Area Under the Curvecalculated at the last experimental point) for Box A wild type, Box Avariant number 64, PEGylated Box A wild type and PEGylated Box A variantnumber 64 curves are reported.

TABLE 2 Calculated AUC_(last) data for Box A wild type (WT), Box Avariant number 64 (64), PEGylated Box A wild type (WT) and PEGylated BoxA variant number 64 (PEG-64) curves. AUC_(last) (μM*min) WT 8.899 6414.97 PEG-WT 275.5 PEG-64 332.53.6 Discussion

The relative gain in AUC_(last) conferred to WT by the mutation is 1.68×(WT vs. 64), most likely due to the higher protease resistance in thesub cute compartment. The relative gain in AUC_(last) conferred to WT byPEGylation is 31× (WT vs. PEG-WT), mainly due to impaired renalfiltration of the PEG conjugate, but also to protection from proteaseaction. Putting together mutation and PEGylation yields a relative gainin AUC_(last) of 37× (WT vs. PEG-64). Unexpectedly, the twomodifications together have a positive effect on animal exposure to theprotein that is superior to the sum of the contributions of the singlemodifications (i.e., 37×>1.68×+31×). Thus, single point mutation andPEGylation have a synergistic, cooperative effect on the pharmacokineticprofile of the native protein. A possible explanation of this phenomenoncould be that PEGylated proteins are not completely protected fromproteolysis in the sub cute compartment. The introduction of a singlepoint mutation gives a boost to resistance in this compartment, allowinghigher quantities of protein to enter blood circulation and to be thenprotected from renal filtration by the bulky PEG chain.

The invention claimed is:
 1. A method for selectively inhibiting and/orantagonizing extracellular HMGB1 activity in a patient, comprisingadministering to a patient in need of such treatment, an effectiveamount of a polymer conjugate of the human and/or non human wild typeHMGB-1 high affinity binding domain Box-A (HMGB1 Box-A) selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:117, and SEQ ID NO:223,or a polypeptide variant of the human and/or non human HMGB-1 Box-A,which has an antagonist activity with respect to the pro-inflammatoryactivity of the HMGB1 Box-B or the full length HMGB1 wildtype protein,wherein the amino acid sequence of said polypeptide variant is selectedfrom the group consisting of SEQ ID NO(s): 33, 35, 37-39, 42-45, 47-49,52, 55, 57, 59, 62, 64, 67, 69, and
 104. 2. The method of claim 1,wherein the amino acid sequence of said polypeptide variant of the humanHMGB-1 Box-A is defined by SEQ ID NO:
 64. 3. The method of claim 1,wherein the polymer is a linear or branched polymer moiety selected frompolyethylene glycol (PEG) or methoxy-polyethylene glycol (m-PEG).
 4. Themethod of claim 1, wherein said patient is suffering from an HMGB-1associated pathological condition mediated by activation of theinflammatory cytokine cascade, wherein said HMGB-1 associatedpathological condition is selected from the group consisting ofpsoriasis, rheumatoid arthritis, epilepsy, neonatal hypoxia,endotoxemia, and sepsis.
 5. The method of claim 4, wherein saidautoimmune disease is rheumatoid arthritis.
 6. The method of claim 4,wherein said systemic inflammatory response syndrome is sepsis orendotoxemia.
 7. The method of claim 1, wherein said polymer isadministered in combination with a further agent capable of inhibitingan early mediator of the inflammatory cytokine cascade.
 8. The method ofclaim 7; wherein said further agent is an antagonist or inhibitor of acytokine, an antibody to RAGE, a nucleic acid or nucleic acid analoguecapable of inhibiting RAGE expression, a small synthetic moleculeantagonist of the HMGB-1 interaction with RAGE or soluble RAGE (sRAGE);the N-terminal lectin-like domain (D1) of native or mutatedthrombomodulin, a synthetic double-stranded nucleic acid or nucleic acidanalogue molecule with a bent, shape structure, K-252a, a salt orderivative of K-252a, a polymer conjugate of K252a, a derivative ofK-252a polymer conjugate, or an inhibitor of the interaction HMGB-1 withat least one Toll-like receptor (TLR).
 9. The method of claim 8 whereinsaid at least one Toll-like receptor (TLR) is selected from the groupconsisting of TLR2, TLR4, TLR7, TLR8 and TLR9.
 10. The method of claim8, wherein said inhibitor of the interaction of HMGB-1 with at least oneToll-like receptor (TLR) is a monoclonal or polyclonal antibody anucleic acid or nucleic acid analogue capable of inhibiting TLRexpression; or a synthetic molecule having a size of less than 1000Dalton.
 11. The method of claim 8, wherein said antagonist or inhibitorof an cytokine is selected from the group consisting of TNF, IL-1α,IL-1β, IL-R_(a), IL-6, IL-8; IL-10, IL-13, IL-18, IFNγ, MIP-α, MIF-1β,MIP-2, MIF and PAF.
 12. The method according to claim 1, wherein saidpolymer conjugate of the human and/or nonhuman wild type HMGB-1 highaffinity binding domain Box-A (HMGB1 Box-A) has increased watersolubility, improved pharmaceutical manageability, improvedpharmacokinetic and/or bioavailability, increased protease resistance, alonger half-life in body fluids, and decreased toxicity and/orimmunogenicity as compared to wildtype HMGB1 Box A protein.
 13. A methodfor antagonizing the pro-inflammatory activity of a HMGB1 Box B or thefull length HMGB1 wildtype protein comprising administering to a patientin need of such treatment, an effective amount of a polymer conjugate ofthe human and/or nonhuman wild type HMGB-1 high affinity binding domainBox-A (HMGB1 Box-A) according to SEQ ID NO:1, or a polypeptide variantof the human and/or non human HMGB-1 Box-A, which has an antagonistactivity with respect to the pro-inflammatory activity of the HMGB1Box-B or the full length HMGB1 wildtype protein, wherein the amino acidsequence of said polypeptide variant is selected from the groupconsisting of SEQ ID NO(s): 33, 35, 37-39, 42-45, 47-49, 52, 55, 57, 59,62, 64, 67, 69, and 104.